In-Vitro Inhibition of Pythium Ultimum, Fusarium Graminearum, And

agronomy Article In-Vitro Inhibition of Pythium ultimum, Fusarium graminearum, and Rhizoctonia solani by a Stabilized Lactoperoxidase System alone and in Combination with Synthetic Fungicides Zachariah R. Hansen, Marie K. Donnelly and Stéphane Corgié * Zymtronix Catalytic Systems Inc., Kevin M. McGovern Center for Venture Development in the Life Sciences, Cornell University, Ithaca, NY 14853, USA; [email protected] (Z.R.H.); [email protected] (M.K.D.) * Correspondence: [email protected]; Tel.: +1-607-351-2639 Received: 30 October 2017; Accepted: 21 November 2017; Published: 23 November 2017 Abstract: Advances in enzyme stabilization and immobilization make the use of enzymes for industrial applications increasingly feasible. The lactoperoxidase (LPO) system is a naturally occurring enzyme system with known antimicrobial activity. Stabilized LPO and glucose oxidase (GOx) enzymes were combined with glucose, potassium iodide, and ammonium thiocyanate to create an anti-fungal formulation, which inhibited in-vitro growth of the plant pathogenic oomycete Pythium ultimum, and the plant pathogenic fungi Fusarium graminearum and Rhizoctonia solani. Pythium ultimum was more sensitive than F. graminearum and R. solani, and was killed at LPO and GOx concentrations of 20 nM and 26 nM, respectively. Rhizoctonia solani and F. graminearum were 70% to 80% inhibited by LPO and GOx concentrations of 242 nM and 315 nM, respectively. The enzyme system was tested for compatibility with five commercial fungicides as co-treatments. The majority of enzyme + fungicide co-treatments resulted in additive activity. Synergism ranging from 7% to 36% above the expected additive activity was observed when P. ultimum was exposed to the enzyme system combined with Daconil® (active ingredient (AI): chlorothalonil 29.6%, GardenTech, Lexington, KY, USA), tea tree oil, and mancozeb at select fungicide concentrations. Antagonism was observed when the enzyme system was combined with Tilt® (AI: propiconazole 41.8%, Syngenta, Basel, Switzerland) at one fungicide concentration, resulting in activity 24% below the expected additive activity at that concentration. Keywords: Pythium; Fusarium; Rhizoctonia; enzyme stabilization; lactoperoxidase; glucose oxidase; fungicide 1. Introduction Fungicides are critical for the control of many economically important plant diseases and have been an integral component of crop production for decades [1–3]. All fungicides impose a selection pressure on their target pathogen populations, and some fungicides are prone to the development of resistance. This is especially true for fungicides with a single target site, as mutations in the target gene can lead to the selection of a resistant sub-population in the pathogen population with fungicide applications [4–7]. Some widely used examples of such fungicides include succinate dehydrogenase inhibitors (SDHIs), quinone outside inhibitors (QoIs), de-methylation inhibitors (DMIs), and phenylamides (PAs). Fungicides with multi-site modes of action are less likely to select for resistance [8]. Managing fungicide resistance has been a priority since it began to appear in the 1960s and 1970s [8]. Concerns over the loss of fungicide efficacy have resulted in the development and implementation of various resistance management strategies. Such strategies include applying mixtures of fungicides with Agronomy 2017, 7, 78; doi:10.3390/agronomy7040078 www.mdpi.com/journal/agronomy Agronomy 2017, 7, 78 2 of 13 different modes of action (often a site-specific with a multi-site), rotating fungicides with different modes of action, restricting the number of applications per season, maintaining the manufacturer’s recommended dose, and implementing cultural control practices as part of an integrated pest management (IPM) program [8]. An important consideration regarding fungicide mixtures is the potential interaction between different fungicides, which may lead to antagonistic or synergistic activity against target pathogens [9–11]. Identifying such interactions is crucial for the effective and efficient deployment of new fungicide chemistries prior to their incorporation into an IPM program. An element of IPM that has gained attention over the past several years is the use of biologically-based pest control solutions, known as biocontrols or biopesticides. Mechanisms of biocontrols and biopesticides include antibiosis, resource competition, and disease resistance-induction [12]. Regarding plant disease management, other than the use of resistant host varieties, biocontrols are considered as living organisms that suppress the activity or reduce populations of plant pathogens. Products that achieve the same outcome, but do not contain living organisms, and are fermented or extracted from natural sources, are considered biopesticides [12]. Enzymes are integral to the antagonistic activity of certain biocontrol microorganisms. Lytic enzymes, such as chitinases, glucanases, and proteases, are secreted by a number of microbes and are known to be suppressive to plant pathogenic fungi and bacteria [13–17]. However, deploying these enzymes directly for plant disease management has received little attention, probably in part due to the costs that are associated with obtaining commercially relevant quantities of enzyme, and subsequently stabilizing the enzymes for industrial use. Advances in the field of biocatalysis, particularly in the areas of protein engineering enzyme immobilization and stabilization, are making the use of enzymes in agriculture increasingly feasible [18–20]. As pesticide resistance and government regulation continue to decrease the number of effective compounds that are available to farmers [8,21], enzymes represent a promising new frontier in crop protection. Enzyme immobilization can be achieved through several approaches, including adsorption of the proteins onto a carrier, protein encapsulation inside a carrier, or cross-linking. Enzyme immobilization increases enzyme stability and has been shown to increase enzyme activity in certain systems [18]. For example, immobilization of horseradish peroxidase by adsorption onto magnetic nanoparticles and the subsequent entrapment by self-assembly increased enzyme activity and reduced inhibition by the H2O2 substrate and reaction products when compared to the free enzyme [22,23]. Maximizing enzyme activity and stability will likely be paramount for the economic viability and, ultimately, the adoption of stabilized enzymes as alternatives to conventional synthetic pesticides by the agriculture industry. Lactoperoxidase (LPO) is an enzyme with antimicrobial properties and occurs naturally in tears, saliva, and milk [24,25]. Lactoperoxidase is extracted from bovine milk and can be obtained relatively simply [26], making it an attractive option for experimentation as well as commercialization. LPO catalyzes the oxidation of thiocyanate and iodide ions to antimicrobial hypothiocyanite (OSCN−) − and hypoiodite (OI ), respectively, in the presence of hydrogen peroxide (H2O2). The H2O2 can be provided by the activity of glucose oxidase (GOx), which is extracted from the fungus Aspergillus niger, on β-D-glucose in the presence of oxygen. These unstable compounds, in turn, oxidize sulfhydryl groups in the cell membranes of microbes, leading to the inhibition of glucose transport, glycolysis, respiration, and ultimately cell death [24]. The LPO system, which here refers to LPO, GOx, and substrates, is known to inhibit Gram positive and Gram negative bacteria, as well as fungi [27]. − − Furthermore, once OSCN and OI are depleted, H2O2 accumulates through the activity of GOx which leads to additional oxidative stress on microbial cells [28]. The objectives of this study were to (i) test the ability of a stabilized LPO and GOx system to inhibit the growth of P. ultimum, F. graminearum, and R. solani in vitro; and, (ii) determine if the combined effect of stabilized LPO and GOx with five commercial fungicides resulted in synergistic, additive, or antagonistic activity when compared to each active ingredient alone. Agronomy 2017, 7, 78 3 of 13 2. Results 2.1. Optimizing the Stabilized Enzyme Formulation P. ultimum, F. graminearum, and R. solani all showed sensitivity, to varying degrees, to the stabilized enzyme formulation in preliminary tests. As a result, an experiment was conducted to identify enzyme concentrations that would result in a measurable reduction in fungal growth without being completely inhibitive. P. ultimum showed greater sensitivity to the enzyme formulation than F. graminearum and R. solani in preliminary experiments so P. ultimum was tested at lower enzyme concentrations than F. graminearum and R. solani for formula optimization (Table1). The three highest enzyme concentrations tested on P. ultimum resulted in 100% inhibition. The lowest concentration tested resulted in 31% inhibition (Table1). All three of the enzyme concentrations that were tested on F. graminearum and R. solani resulted in reduced growth, and ranged from 74% to 57% growth reduction for F. graminearum and 72% to 63% growth reduction for R. solani (Table1). Based on these results, the lowest concentration tested was chosen for P. ultimum (4.0 nM LPO and 5.2 nM GOx) and F. graminearum and R. solani (119.0 nM LPO and 154.7 nM GOx) fungicide experiments. The stabilized enzyme formulation was prepared as carboxymethyl cellulose (CMC) film disks at these concentrations for the fungicide synergy experiments. Table 1. Enzyme concentrations used to optimize formulations

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